Myosin II ATPase is an enzyme associated with the contraction process of smooth muscle. By genetic and chromatographic methods, there are presently four Myosin II ATPase isoforms that have been detected in smooth muscle. The differences in their polypeptide sequences and differences in their conformational structures have unknown consequences. Isoforms of a protein can be produced by related genes, or may arise from the same gene by alternative splicing. Some isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic differences between alleles of the same gene. SNPs can occur at specific individual nucleotide positions within a gene. In smooth muscle there are at least four slice variants from a single Myosin ATPase gene. In addition to the heavy chains there are 2 light chains and at least four splice variants of the light chains (Aguilar, H. N.: Xiao, S: Knoll, A. H.; Yuan, X (2010) “Physiological pathways and molecular mechanisms regulating uterine contractility” Human Reproduction Update: 16 (6) 725).
Blebbistatin is a small organic molecule with chemical structure name: 3a-hydroxy-6-methyl-1-phenyl-1, 2, 3, 3a-tetrahydro-4H-pyrrolo[2,3b]-quinolin-4-one, that was discovered by vitro testing for an inhibitor of a non-muscle Myosin II ATPase using IC50 measurements as a criteria (Straight A F, Cheung A, Limouze J et al. “Dissecting temporal and spatial control of cytokinesis with a Myosin II Inhibitor” Science 2003; 299: 1743-7).

An IC50 measurement is a determination of the concentration of an inhibitor that causes a 50% inhibition of a biological process such as an enzyme activity, and the IC50 can have units of concentration, such as uM (micromolar). The potency of an inhibitor is inversely-related to its IC50 value. The Myosin II ATPase inhibitor, blebbistatin, has been reported to have significantly different IC50 values in vitro in different tissues, such as for example, in rabbit striated skeletal muscle bebbistatin inhibits the Myosin II ATPase with an IC50 value of 0.5 uM; in pig cardiac muscle bebbistatin inhibits the Myosin II ATPase with an IC50 value of 1.2 uM); and in turkey smooth muscle bebbistatin inhibits the Myosin II ATPase with an IC50 value of 79.6 uM). (Limouze J, Straight A F, Mitchison T, Sellers J R. “Specificity of blebbistatin, an inhibitor of Myosin II.” J Muscle Res Cell Motil 2004; 25:337-41). Furthermore, Ekman et al. reported that 10 uM blebbistatin did not block an adult mouse bladder smooth muscle contraction when the tissue is depolarized using potassium chloride. However, 10 uM blebbistatin did inhibit the bladder contraction of a newborn mouse. The new-born mouse bladder was found to predominantly express a different Myosin II ATPase isoform known as the non-muscle Myosin II ATPase isoform (Ekman M, Fagher K, Wede M, Stakeberg K, Arner A. “Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice” J Gen Physiol 2005; 125: 187-96).
J. Allingham et al. (2005), based on X-ray diffraction data, proposed a model for specific inhibitory binding of blebbistatin to the Dictyostelium discoideum (slime mold-soil amoeba) Myosin II ATPase. This model proposes that the binding of blebbistatin to the ATPase depends upon multiple hydrophobic, ionic, and hydrogen bonding interactions.
Allingham's model focuses on the interaction of the blebbistatin molecule with four specific amino acid residues of the ATPase: Ser456, Thr474, Tyr634, and Gln637. The model proposes that the blebbistatin IC50 for a Myosin II ATPase isoform strongly correlates with the extent of amino acid residue homology at positions 456, 474, 634, and 637 of the Dictyostelium discoideum ATPase. Given this model's focus on these four amino acid positions, and that Non-muscle Myosin IIA ATPase and Smooth Muscle Myosin II ATPase have the same four “interacting” amino acids: Ala456, Thr474, Tyr634 and Gln637, then the blebbistatin IC50 for these two ATPases should be similar. However, this is not the case; the IC50's are not similar. Blebbistatin's IC50 is 5.1 uM for inhibition of the Non-muscle Myosin IIA ATPase and 79.6 uM for inhibition of Smooth Muscle Myosin II ATPase. Allingham et al. admitted that their model for blebbistatin's binding affinity can be influended by “second sphere interactions”. More simply stated, the structure activity relationship (SAR) of blebbistatin for one particular Myosin II ATPase isoform does not allow one to predict the SAR of blebbistatin for another Myosin II ATPase isoform.
J. Toth (2006) tested 14 blebbistatin derivatives at a single concentration of 100 micromolar to screen for their potency as a potential inhibitor of the following Myosin ATPases: (1) Chicken Skeletal Myosin II ATPase, (2) Porcine Cardiac Muscle Myosin II ATPase, (3) Scallop Striated Muscle Myosin II ATPase, (4) Chicken Gizzard Smooth Muscle Myosin II ATPase, (5) Human Non-Muscle Myosin IIA ATPase, (6) Human Non-Muscle Myosin IIB ATPase, (7) Mouse Non-Muscle Myosin IIC ATPase, (8) Mouse Myosin Va ATPase, and (9) Human Myosin X ATPase. A blebbistatin derivative has a blebbistatin core with a selected substituent group. Toth's numbering system for the carbon atoms of the blebbistatin molecule (taken from FIG. 46, pg 58 of her PhD thesis) is displayed below:

Toth tested blebbistatin derivatives (separately tested (+) and (−) enantiomers) with a 3-methyl, a 4-methyl, a 5-methyl, a 5-hydro, and a 6-methyl. Also tested were a 7-hydroxyl-5-hydro, a 4-nitro, a 5-bromo, and an 8-fluoro.
Toth's screening tests did not find any blebbistatin derivatives that are a more potent inhibitor than blebbistatin. Toth found (−) blebbistatin and 6-methyl derivatives inhibited cardiac muscle Myosin ATPase (Toth table IV, page 94). Toth also found that (−) blebbistatin, 5-bromo-, 6-methyl, 4-methyl and 4-nitro blebbistatins inhibited Non-muscle Myosin ATPases (Toth table IV, page 94).
C. Lawson (2011) devised a novel synthetic route to make blebbistatin derivatives from a common chemical intermediate but Lawson's method has extra chemical reaction steps compared to the conventional blebbistatin synthesis route. Lawson made 11 derivatives of blebbistatin from the common chemical intermediate wherein the substituent at the 1 position of the blebbistatin was varied. The 1 position substituents were 4-methoxyphenyl, 4-methylphenyl, 4-cyanophenyl, 4-nitrophenyl, 4-chlorophenyl, 3-chlorophenyl, 3-trifluoromethylphenyl, thienyl, pyridyl, 4-phenylphenyl, and 4-iodophenyl. Lawson did not disclose any biological test results concerning these 11 blebbistatin derivations.
Kepiro et al. (2014) made and tested four blebbistatin derivatives with a 1-position substitutent, namely the 4 chlorophenyl, the 4-iodophenyl, the 4-nitrophenyl, and the 4-azidophenyl-derivative of blebbistatin. In the same research group, Varkuti (2016) synthesized and tested a blebbistatin derivative with a 4-aminophenyl substituent. Interestingly Kepiro's blebbistatin derivative with a 1-(4-azidophenyl) substituent forms a covalent bond somewhere with the Myosin II ATPase which inhibits the ATPase.
Surprisingly, it has been more than about 12 years since Straight published the invention of blebbistatin, there have been no reports of a more potent reversible-inhibitor blebbistatin derivatives. There is a need for reversible inhibitor blebbistatin compounds which are more potent than blebbistatin. In addition there is a need for a structure activity relationship (SAR) for the derivatives of reversible inhibitor blebbistatin that inhibit Myosin II ATPase.
The treatment of overactive bladder with blebbistatin and with blebbistatin derivatives is a significant medical invention (see published US Patent Appln. No. US2014/0200238). Lower urinary tract (LUT) dysfunction can include an overactive bladder (OAB). The bladder's smooth muscle is referred to as the detrusor muscle. The main symptoms of an overactive bladder are an increased day-time and night-time frequency of urination, an increased frequency of the urge to urinate and a reduced ability to control urination. OAB is increasing in the ageing population and there are few highly-effective or tolerable treatments (Kelleher C J, Kreder K J, Pleil A M, Burgess S M, Reese P R, “Health-related quality of life of patients receiving extended-release tolterodine for overactive bladder” Am J Manag. Care 2002; 8: S608-15).
A cause of OAB may be a frequent or an excessive release of acetylcholine from cholinergic nerves innervating or terminating near the bladder smooth muscle. A common pharmaceutical therapy for OAB is administration of an anti-muscarinic drug to block the excessive acetylcholine from binding to acetylcholine receptors. Unfortunately, such anti-muscarinic therapy has only a 65-75% efficacy in treating major symptoms of overactive bladder (OAB) in a patient, and its use is limited due to drug side effects such as blurred vision, dry mouth and constipation. (Zhang, X, Kuppam, D S R, Melman, A, DiSanto, M E. “In vitro and In vivo relaxation of urinary bladder smooth muscle by the selective Myosin II inhibitor, blebbistatin” BJU Internat., 2010 e-publication and 2011 journal publication; Volume 107, Issue 2, pages 310-317). About 75% of the OAB patients discontinue taking an anti-muscarinic drug to treat OAB because the patient finds such side effects intolerable.
Various types of drugs for a treatment of OAB include using α-adrenergic antagonists, β-adrenoceptor agonists, membrane channel activity modulators, phosphodiesterase inhibitors, and prostaglandin-synthesis inhibitors (Andersson K E, Chapple C R, Cardozo L et al. “Pharmacological treatment of overactive bladder: report from the International Consultation on Incontinence” Curr Opin Urol 2009; 19: 380-94). Some pharmaceutical drug therapies for OAB modify the myogenic pathway of the bladder's smooth muscle (detrusor muscle) (Yoshimura N, Kaiho Y, Miyazato M et al. “Therapeutic receptor targets for lower urinary tract dysfunction.” Naunyn Schmiedebergs Arch Pharmacol 2008; 377: 437-48). Myogenic pathways are believed to be an important trigger for detrusor muscle contraction and relaxation cycling. In smooth muscle there are changes in cell membrane tension during the muscle contraction and relaxation cycle. The stretch declines during muscle contraction and increases during muscle relaxation. Increased membrane tension opens membrane ion channels whose conductance depolarizes the cell transmembrane potential. The change in transmembrane potential increases cytoplasmic calcium ion levels and other biochemical processes which stimulate muscle contraction. Contraction relieves membrane tension and the processes reverse during muscle relaxation when membrane tension has again increased due to muscle lengthening. Different smooth muscle organs vary in degree, frequency and time profile of their contractions. The response of different smooth muscles to drugs is unpredictable. It is appreciated that there is serial and parallel biochemical signal processing and often with positive and negative feedback controls. Also, there can be changes in genetic expression of smooth muscle during development and aging. Endocrine and exocrine processes also modulate smooth muscle function.